Quantification of Inflammatory Cytokines in Substantia Nigra

The substantia nigra was homogenized with the protein extraction buffer containing 100 mM Tris-HCl (pH 7.4), 750 mM NaCl (sodium chloride), 10 mM EDTA (ethylenediaminetetraacetic acid), 5 mM EGTA (ethylene glycol tetraacetic acid), and protease inhibitors (Roche, Basel, Switzerland) [5 (link), 45 (link)]. The samples were centrifuged at 1000g for 10 min at 4°C. The supernatant was collected and centrifuged again at 20,000g for 40 min at 4°C to remove remaining debris. ELISA was performed using a Milliplex MAP Rat cytokine/chemokine magnetic bead panel kit according to the provider's protocol (RECYTMAG_65K; Millipore, Temecula, CA, USA), and reading was made by using the LUMINEX MAGPIX detection system with xPONET software (Millipore Corporation, Billerica, MA, USA). The sensitivity ranges were 2.4 to 10,000 pg/mL for TNF-α and IL-1β, 73.2 to 300,000 pg/mL for IL-6, and 7.3 to 30,000 pg/mL to IL-4 and IL-10.